Fluorescent dyes as functional molecules have been widely used in various fields of science and technology. Studies on them in life science, clinical diagnosis, immunological assays and etc become the focus of attention throughout the whole world. At present, phenanthridines (EB, PI), acridines (AO), imidazoles (Hoechst, DAPI), cyanine dyes (Cy, TOTO, SYTO) and other commercially available fluorescent dyes have played significant role in the fields of genomics, quantitative detection of nucleic acids, blood cell analysis and etc. However, all these commercially available fluorescent dyes have their own application limitations. The major problem is many of them are restricted to fixed cell samples, such as TOPRO, the TOTO dye family, ethidium bromide (EB), and propidium iodide (PI), which require membrane permeabilization or similar method to label the DNA efficiently. However, fixation of cells often produces undesired artifacts [S. Kozubek, E. Lukasova, J. Amrichova, M. Kozubek, A. Liskova, J. Slotova, Anal. Biochem. 2000, 282, 29-38]. At the same time, acridine and phenanthridine dyes such as EB are toxic and carcinogenic. Secondly, there are a considerable number of fluorescent dyes need ultraviolet illumination as excitation light source, such as DNA specific crescent-shaped dyes 4′,6-diamidino-2-phenylindole (DAPI), Hoechst33258, Hoechst34580, which emit blue fluorescence upon binding to DNA. As the UV light damages cellular DNA, protein and other components, the use of them is very restricted in time [S. K. Davis, C. J. Bardeen, Photochem. Photobiol. 2003, 77, 675-679]. Besides, it's difficult for excitation light to penetrate into the inside biological tissues due to the strong absorption of ultraviolet light by some components in biological samples. Furthermore, the self fluorescence of some biological components may lead to a high fluorescence background that interferes with detection. Therefore, exploring novel fluorescent dyes meeting the multiple criteria of excellent spectral properties, low toxicity and live cell permeability is still the key to promote the development of fluorescent analysis technology, life science and other fields.
Among various sorts of fluorescent dyes, cyanine dyes with their wide wavelength emission scope, large molar extinction coefficient and proper fluorescence quantum yield, are extensively applied as biofluorescent probes, CD or VCD recording materials, photosensitive agents for photosensitive materials, photoelectric conversion materials, etc. Quinoline asymmetric cyanine dyes stand out from all cyanine classes in the applications of areas such as genomics, nucleic acids quantitative detection, blood cell analysis and etc because of their specificity showing high affinity with nucleic acids and basically no association with other biomacromolecules. The interaction modes of these asymmetric cyanine dyes with nucleic acids are intercalation to base pairs, groove binding and electrostatic attraction. Specific binding mode and binding affinity depends on the structure of the dyes and the ratio of dyes to base pairs. A typical type of asymmetric cyanine dyes are TOTO and its analogues and derivatives thereof. TOTO (thiazole orange dimer) and YOYO (oxazole orange dimer), developed by Glazer research group, are multicationic asymmetric cyanine fluorescent dyes having a high affinity to nucleic acids, of which different heterodimeric analogues and derivatives can be obtained by changing the dye molecules on the ends of the polymethine bridge. These dyes show no fluorescence in solution, which reduces the interference from the fluorescence background during detection, but do upon binding to nucleic acids. Jason et al explored the double intercalation of TOTO and YOYO into DNA using solution viscosity measurements and atomic force microscopy [J. A. Bordelon, K. J. Feierabend, S. A. Siddiqui, L. L. Wright, J. T. Petty, J. Phys. Chem. B 2002, 106, 4838-4843]. Fürstenberg et al further expounded the dynamic mechanism of the fluorescence enhancement using ultrafast fluorescence up-conversion and time-correlated single photon counting technology [A. Fürstenberg, M. D. Julliard, T. G. Deligeorgiec, N. I. Gadjev, J. Am. Chem. Soc. 2006, 128, 7661-7669]. Many of this type of asymmetric cyanine dyes are commercially available, such as SYTOX, TOTO, POPO, BOBO, YO-PRO and etc. But such commercially available dyes usually have large molecular weight and complex structures, which are live cell impermeant and can only be utilized to nucleic acids detection in vitro.